Abstract Caloric restriction (CR) extends life/healthspan in multiple species, although one drawback of CR is poor compliance. This has led to alternative dietary approaches aimed at healthspan extension. We have established an isocaloric twice-a-day (ITAD) feeding intervention wherein food is partitioned into two feeding intervals each day, without CR. ITAD mice are fed at two 2-hour intervals each day such that total calories consumed are similar to those eaten by ad-libitum (Ad-lib) controls in 24 hours. Despite absence of CR, ITAD-fed mice are protected against diabetes and metabolic syndrome of aging. The mechanisms by which ITAD feeding mediates these benefits remain unknown. Circadian-wide analyses revealed that ITAD feeding causes marked temporal shifts in patterns of the quality control pathway autophagy. Surprisingly, ITAD mice show stimulation of autophagy flux immediately after the 1st feeding window at 11am and marked suppression at 7pm. While autophagy activation at 11am increases fat utilization, suppression of autophagy at 7pm associates with inhibition of gluconeogenic gene expression and glucose production in aging mice. The mechanism by which ITAD feeding suppresses glucose production is not known. Our recently published work has shown that core clock proteins are targets for lysosomal degradation, and that autophagy specifically degrades CRY1. We have found that autophagy is stimulated from 3pm to 7pm in livers from young Ad-lib-fed mice, leading to the timed- degradation of CRY1a robust inhibitor of gluconeogenesis. CRY1 binds to the autophagosome marker LC3 through its LC3-Interacting Region (LIR) motifs. Acute autophagy blockage or inactivation of its LIR motifs promotes CRY1 accumulation. Since CRY1 suppresses gluconeogenesis, its accumulation blocks gluconeogenesis and lowers blood glucose. Interestingly, our preliminary data reveal that aged and obese mice each display accelerated degradation of CRY1 by autophagy, leading to sustained gluconeogenesis and hyperglycemia. Although autophagy flux typically decreases with age, we propose that age-related hyperphosphorylation of CRY1, a modification shown to trigger its degradation, favors its sequestration and rapid degradation by residual autophagy. From an interventional stand-point, our preliminary studies indicate that ITAD feeding prevents the loss of CRY1 levels at 7pm by suppressing autophagy at this time-frame. We propose that approaches preventing the age-related decline in CRY1 protein will normalize blood glucose levels and prevent diabetes. On this premise, we hypothesize that age-related hyperphosphorylation of the core circadian repressor CRY1 accelerates its autophagic degradation leading to hyperglycemia and type 2 diabetes. We propose the use of two interventions to maintain CRY1 levels in aged mice, i.e., ITAD feeding, and small molecules designed to mask LIR motifs of CRY1 and block its degradation that, in turn, will suppress gluconeogenesis and prevent age-related diabetes. In this proposal, we will: (1) Determine the age and dietary- stress-related changes in protein levels and lysosomal degradation rates of CRY1 and additional core circadian proteins in liver; (2) Determine how age-related modifications to CRY1 accelerates its degradation and alters gluconeogenesis; (3) Determine whether ITAD feeding reverses age-associated diabetes by increasing CRY1 levels, and (4) Identify small molecule inhibitors to block CRY1-LC3 interaction with potency, selectivity and favorable drug-like properties and prevent hyperglycemia is aged mice. Significance: Age-related metabolic syndrome/type 2 diabetes is a significant health problem in the United States that increases sharply to ~44% in individuals older than 50 years. Type 2 diabetes impacts healthspan through effects on cardiovascular and cerebrovascular systems, physical activity, vision, and cognition. Type 2 diabetes is thought to predispose to neurodegenerative disease, including Alzheimer?s dementia. 1